Project Summary: Macrophages are key effector cells of the immune system, with critical functions in killing of microbes, production of inflammatory regulators, and tissue repair. However, an excessive macrophage response contributes to the pathology of cancer as well as inflammatory and degenerative diseases. In addition, unchecked proliferation of macrophage precursors in lieu of differentiation leads to acute myeloid leukemia. To better address how to modulate macrophage function to help abate diseases that involve changes in macrophage biology, we must understand the critical molecular pathways that govern macrophage differentiation and regulate their activity. We propose that one of these pathways will involve mRNA polyadenylation, an essential maturation step in which mRNA precursor is trimmed at its 3' end and a poly(A) tail (pA) added. Changing the position of the pA site through a process called alternative polyadenylation (APA) plays an important, increasingly appreciated role in regulation of gene expression. Shortening of the 3' untranslated region can remove regulatory sequences that control RNA stability, translation, and subcellular localization, whereas coding region shortening can dramatically alter protein function. While global changes in APA have been observed in tumor progression and other types of cellular differentiation, the contribution of APA to macrophage differentiation has not been studied. We hypothesize that a global shift in APA is required for macrophage differentiation and that this shift is driven by changing levels of APA regulators. Our objective is to determine how APA contributes to macrophage differentiation, with the long-range goal of defining how this might be manipulated in therapeutic settings to promote differentiation and modulate macrophage function. Our specific aims will 1) determine the global pattern of APA during macrophage differentiation, the functional classes of genes impacted by APA, and sequence features that might characterize these sites, 2) define drivers of macrophage APA and the consequence that altering their expression has on differentiation as well as well-characterized macrophage functions such as cytokine production, migration, and phagocytosis, and 3) determine the molecular mechanisms that alter the levels of the proteins that regulate APA. Because macrophage are a first line of defense for many diseases and dysregulation of their differentiation leads to leukemias, our proposed studies should ultimately inform new therapeutic tools to modulate macrophage production. They will also broadly advance our understanding of general and tissue-specific APA paradigms. s